CREB (cAMP response element binding protein) is a protein member of the bZIP subfamily of transcription factors. Activation of CREB occurs via phosphorilation of a single serine residue. Once phosphorylated, CREB dimmers, bind to specific CRE (cAMP response element) sites on target genes and regulates gene expression. CREB is of particular interest in drug addiction because its activation is downstream of the cAMP-signaling pathway, whose upregulation has been extensively characterized as an adaptation to chronic exposure to drugs of abuse (Chao and Nestler 2004). Upregulation of the cAMP pathway and activation of CREB seems to be crucial for the effects of drugs on the brain reward and motivational systems, such as the nucleus accumbens, ventral tegmental area, amygdala and frontal cortex, to name a few. In several of these regions, activation of CREB occurs in response to acute and chronic administration of different drugs of abuse, such as opiates, stimulants and alcohol. The regulation of CREB phosphorylation and the function of CREB in addiction vary with respect to multiple parameters, including the identity of the substance (opiates versus cocaine), the nature of the exposure (acute versus chronic), and the CNS region or neuronal pathways involved (Nestler 2004). A leading hypothesis is that drug-induced activation of CREB in these motivation centers of the brain underlies some of the common core features of drug addiction seen clinically, by inducing gene expression that leads to permanent molecular changes or drug induced plasticity. Kandel and collaborators were able to demonstrate that inactivation of CREB in hippocampal CA1 neurons impairs learning in the Morris water maze by interfering with some forms of long-term memory formation (Pittenger et al., 2002). To perturb CREB function, a transgenic mouse that expresses KCREB (a mutant of human CREB that is a potent dominant-negative inhibitor) through a forebrain specific promoter was generated. In accordance, over expression of CREB in CA1 neurons facilitated the establishment of long-lasting LTP in hippocampal slices (Barco et al., 2002). Interestingly, recent studies by Bonci and collaborators have shown that a single exposure to cocaine induces long-term potentiation in dopamine neurons (Ungless et al., 2001), a mechanism that, as in memory formation, may contribute to synaptic plasticity and permanent neuronal changes, which may progressively lead to drug addiction. Whether CREB affects the formation of LTP in dopaminergic cells remains to be investigated. Also, Nestler and collaborators have shown that over expression of a dominant negative mutant CREB in the area of the nucleus accumbens (through the neuron specific-enolase promoter) increases the rewarding effects of cocaine, possibly by regulating dynorphin expression. As CREB is a molecular component of ubiquitous nature in the mesolimbic dopamine system, it would be advantageous if we could dissociate its effects by conditionally inactivating its function in separate sets of mesolimbic neurons, one set at the time. This approach would allow us to better understand the specific roles that CREB plays in addiction and other conditions affecting the mesolimbic system.[unreadable] [unreadable] In our laboratory, we are studying genetically modified mice containing a tetracycline inducible dominant inhibitor CREB gene (KCREB) under the control of specific endogenous neuronal promoters. By using a system for temporal, spatial and cell-type specific control of gene expression, KCREB will be induced only in neurons expressing either the dopamine transporter (DAT), the dopamine receptor 1 (Drd1), or 2 (Drd2) and only after treatment with doxycycline (a tetracycline analog). These different knock-in mice will help us determine the functions that CREB mediates during addiction in specific neuronal types of the mesolimbic dopamine system. These conditional animals are being developed with the use of a single DNA construct that has been tested in vitro (see Bdckman et al., 2004). We will first determine if the genetic modifications introduced in this new animal strain produce the expected phenotype. Upon verification of a biologically functional mutation, these animals will be used for further in depth studies of CREB function during drug addiction. Exhausting behavioral, morphological and functional studies of these animals will generate data that correlates CREB inactivation in D1-, D2- or DAT-positive neurons to addiction.[unreadable] [unreadable] We have generated all the strains described above and are currently under study.